Since nitric oxide (NO) was reported to be an endothelium-derived relaxing factor in 1987, its various roles as a physiologically active substance have been found. NO is synthesized from L-arginine as a substrate by NO synthase (NOS), and controls a wide variety of biological responses not only in cardiovascular systems but also in immune systems and central nervous system, in which NO is involved in bio-defense response through macrophage or in acquisition of synaptic plasticity in memory and learning. It is also considered that NO relates to some diseases including arteriosclerosis, cerebral apoplexy and hypertension in cardiovascular systems, infectious diseases in immune systems, and dementia and Alzheimer's disease in central nervous system.
NO is an unstable and short-life molecule labile to oxidation with enzymes or reactive oxygen species in living body. So, it is difficult to detect NO at a physiological concentration, and therefore the dynamics of NO in living body is in an unknown area.
A method for detection of NO is a fluorescein derivative having a diamino group reacting with NO (diamino-fluoresceins; DAF) (Non-Patent document 1). Though DAF is not fluorescent in the absence of NO, it becomes to triazole to emit green fluorescence upon reacting with NO in the presence of dioxygen. Therefore, NO generation in a cell can be visualized with a fluorescence microscope. With a similar principle, a rhodamine derivative (DAR) emitting red fluorescence has also been reported (Non-Patent document 2).
There was a problem in the prior art, however, that these organic molecules easily accumulate in lipid membranes of cells and emit fluorescence signals there in an NO-independent manner thereby interfering with the detection of low concentration of NO in living cells. In addition, these organic molecules irreversibly react with NO, resulting in causing another problem that the complex dynamics of NO in living body such as a change of NO concentration cannot be followed.
The invention of this application was made to solve the above-mentioned problems in the prior art, and the purpose of the invention is to provide a new procedure for visualizing the change of NO concentration in the nM order precisely, conveniently and reversibly.